Protease

ABSTRACT

Novel alkaline serine proteases produced by Nocardiopsis sp. strains, for example by aerobic cultivation of strain 10R (NRRL-18262) and Nocardiopsis dassonvillei strain M58-1 (NRRL 18133), the proteases being characterized by a pH optimum of about 8, and by retention of at least 60% of maximum activity at pH 7-11 measured with casein as substrate. 
     The proteases of the invention may be distinguished from the known protease of the Nocardiopsis strain ZIMET 43647 (of East German 2004328) and known Bacillus and fungal alkaline proteases. The Alkaline proteases of strains 10R and M58-1 may also be distinguished from each other. 
     The protease preparations of this invention are useful detergent additives for cold water laundering.

This invention relates to a protease-producing strain of Nocardiopsis,to a process for preparing alkaline protease, to an alkaline proteasepreparation derived from Nocardiopsis, to a detergent additive and to anenzymatic detergent additive.

BACKGROUND OF THE INVENTION

Proteolytic enzymes produced by cultivation of microorganism strainsfrom the genus Bacillus in suitable nutrient media are widely used indetergent compositions. Examples of such commercially availableproteinase products are ALCALASE®, ESPERASE®, SAVINASE®, all supplied byNOVO INDUSTRI A/S, Denmark. These and the similar Bacillus derivedenzyme products from other suppliers are enzymatically active indetergent solutions, at pH values in the range of from 8 to 11 and inthe presence of the sequestering agents, surfactants and bleachingagents normally present in detergent solutions.

The protease in ALCALASE® is produced by cultivating strains of speciesBacillus licheniformis. The proteases in ESPERASE® and SAVINASE® areobtainable by cultivation of alkalophilic Bacillus species, such as thestrains NCIB 10147 and NCIB 10309, respectively. The temperature optimaof the commercially available alkaline proteases is about 60° C.However, these commercial enzymes exhibit a relatively lower activity atroom temperature.

BRIEF STATEMENT OF THE INVENTION

This invention relates to the production, isolation, characterizationand use of novel alkaline proteases obtained from actinomycetemicroorganisms. The proteases of this invention are isolated from theactinomycete Nocardiopsis sp. strain 10R and Nocardiopsis dassonvilleistrain M58-1 which have not been previously known to produce alkalineproteases. The proteases described in this invention are useful asdetergent additives for laundering. The strain ZIMET 43647 of thespecies Nocardiopsis dassonvillei described by GDR patent No. DD 200,4328 is known to produce protease for fibrinolytic andbiological-sludge-clarifying applications. However, the strain was notavailable to the public as of the date hereof, the type strain does notproduce protease, and no other strain of this species is known toproduce protease. Further the aforementioned patented protease has atemperature profile and pH optimum much different from that of proteasesin the invention.

In FIG. 3, the pH profiles for the Nocardiopsis sp. proteases of thisinvention were compared to that reported for the protease from patentstrain ZIMET 43647. Under the same experimental conditions, strains 10Rand M58-1 both have pH optima of 8, while that of the ZIMET 43647protease is 9-10. In addition, the proteases of this invention havebroader pH optima, showing at least 60% of the maximum activity betweenpH 7-11. The ZIMET 43647 protease possesses only 50% of maximum activityat pH 7 and 0% of maximum activity at pH 11. The expression of highproteolytic activity at pH 11 is especially important for detergentapplication.

The temperature profiles for the proteases of this invention werecompared to that reported for the protease from ZIMET 43647 in FIG. 4.Between 40° and 50° C., the ZIMET 43647 protease exhibits 70-100% of itsoptimum activity, which is greater than that shown by the proteases ofthis invention, i.e. 25-65%.

In addition to the aforementioned differences, protease production inthe strains of this invention responds differently to the addition ofmineral salts. The strains of this invention produce the same level ofprotease activity whether or not mineral salts are included in thegrowth medium. The ZIMET 43647 strain is reported to show an improvedresponse upon addition of mineral salts to the growth medium, possiblyindicating the presence of a metalloprotease. As shown clearly in FIG.2b, the protease of this invention has no detectable metalloproteaseactivity.

The Nocardiopsis sp. strain 10R and Nocardiopsis dassonvillei strainM58-1 proteases of this invention are especially effective as detergentadditives at low laundering temperature, e.g. in cool water, say 15°-25°C. compared to the commercially available Bacillus proteases. Inaddition, the Nocardiopsis sp. strain 10R and Nocardiopsis dassonvilleistrain M58-1 proteases of this invention generate protein hydrolysispatterns different from that generated by a Bacillus protease.

Despite many similarities, i.e. pH and temperature optima, stability,pattern of insulin digestion, the proteases from Nocardiopsis sp. strain10R and Nocardiopsis dassonvillei strain M58-1 can be distinguished. Asdescribed in Example V, the molecular weights of the proteases differ(23,500-25,000 for 10R, 20,500 for M58-1). The isoelectric points of thetwo proteases differ as well--pI ≧9.5 for 10R, pI of 9.15 and 8.2 (2bands) for M58-1. In addition, antibodies to the 10R protease indicateonly partial identity to the M58-1 protease and vice versa.

DISCUSSION OF THE INVENTION

There are many current commercial uses for proteases, includingutilization in the detergent industry. The detailed discussion of thenovel Nocardiopsis sp, strain 10R and Nocardiopsis dassonvillei strainM58-1 proteases which follows is limited to usage as a detergentadditive. In addition, the inventors do recognize that other uses forproteolytic enzymes do exist.

It should be recognized that enzymes are affected by the conditions ofusage, including pH, temperature, and for detergent use, the presence ofsequestering agents such as EDTA. In consequence, the Bacillusproteases, such as SAVINASE® which were developed for laundering cottonfabrics at high temperature (60° C.), may not be as effective at thelower wash temperatures (15°-25° C.) recommended for many modern-dayfabrics.

Protease preparations comprising the novel alkaline proteases fromNocardiopsis sp. strain 10R and Nocardiopsis dassonvillei strain M58-1exhibit about 20-50% greater capability for whitening protein-basedstains on cotton (EMPA 116) under simulated laundering conditions at 15°C. than the SAVINASE® protease when used at the same enzyme activitylevel, as evidenced in Table IV.

DETAILED DESCRIPTION OF THE INVENTION

According to a further aspect of this invention there is provided amethod for producing the alkaline proteases, which process ischaracterized by cultivating a protease producing strain of Nocardiopsissp. under aerobic conditions in a nutrient medium containing assimilablesources of carbon, nitrogen, and phosphorus, followed by recovery of theprotease preparation from the fermentation broth.

For further understanding of this invention, reference is made to theattached drawings wherein:

FIGS. 1 and 1a show the elution chromatograms of digestion productsafter 15 minutes and 2.5 hours of treatment of oxidized β-chain ofinsulin with the Bacillus protease product SAVINASE®. FIGS. 1b and 1cshow the elution chromatograms of digestion products after 15 minutesand 2.5 hours of treatment of oxidized β-chain of insulin with theBacillus protease product ALCALASE®. FIGS. 1d and 1e show the elutionchromatograms of digestion products after 15 minutes and 2.5 hours oftreatment of oxidized β-chain of insulin with the protease fromNocardiopsis sp. strain 10R. The protease from Nocardiopsis dassonvilleistrain M58-1 yields digestion products that are similar to those forNocardiopsis sp. strain 10R (data now shown).

FIG. 2 shows the activity of the protease from Nocardiopsis sp. strain10R toward casein as a function of temperature. FIG. 2A shows theactivity of the same protease toward casein as a function of pH. Inaddition, the effects of EDTA (10 mM) and phenylmethyl sulfonyl fluoride(PMSF, 1 mM) on protease activity are also presented. The protease fromNocardiopsis dassonvillei strain M58-1 behaves similarly to the proteasefrom strain 10R in that PMSF, but not EDTA, inhibits activity. Theseresults indicate that the proteases from strains 10R and M58-1 areserine proteases, not metalloproteases.

FIG. 3 shows the differences in pH optima of Nocardiopsis sp. strain10R, Nocardiopsis dassonvillei strain M58-1 and patented strain ZIMET43647.

FIG. 4 presents the differences in temperature optima betweenNocardiopsis sp. strain 10R, Nocardiopsis dassonvillei strain M58-1 andpatented strain ZIMET 43647.

THE MICROORGANISMS

The microorganisms of this invention are aerobic, actinomycete isolatesof Nocardiopsis.

Two strains have been deposited at the Agricultural Research CultureCollection (NRRL), Peoria, US, under the terms of the Budapest Treaty,as follows:

    ______________________________________                                        Depositor's reference                                                                         10R         M58-1                                             Deposit No.     NRRL 18262  NRRL 18133                                        Deposit date    10 Nov. 1987                                                                              13 Nov. 1986                                      Taxonomic designation                                                                         Nocardiopsis sp.                                                                          N. dassonvillei                                   ______________________________________                                    

Mutants and variants of these strains, obtained by methods known in theart, are also within the scope of the invention.

Temperature for growth of both strains is 20° C. to 30° C., no growth ator above 35° C. Optimal pH for growth is 9 and no growth occurs at orbelow pH 8. The aforementioned growth parameters are different fromthose of patented strain ZIMET strain 43647. The ZIMET strain iscultivated at a temperature between 25° and 37° C. (preferably 28° C.)and at an acidity of between pH 6.5 and 7.2.

On Czapek Dox Agar slants mature colonies of Nocardiopsis sp. strain 10Rexhibit gray-green aerial mycelia with black secretions, Nocardiopsisdassonvillei strain M58-1 mature colonies have white to cream coloredaerial mycelia on Czapek Dox Agar slants. The typical Nocardiopsisdassonvillei species have mealy aerial mycelia with a faint yellow-graytint.

The type strain of Nocardiopsis dassonvillei (ATCC 23218) does notelaborate the protease of this invention. It is believed that the typestrain (ATCC 23218) and the Nocardiopsis sp. isolates of this inventionare different variants. Moreover, strain 10R and M58-1 are differentfrom each other by the response to the general protoplasting reagents,egg-white lysozyme and NovoZym™ 234. Strain 10R is unable to be lysed bylysozyme and/or NovoZym™ 234 while strain M58-1 can be protoplasted bylysozyme alone. The typical Nocardiopsis dassonvillei strains aresusceptible to lysozyme (M. C. Shearer et. al., Int. J. Syst. Batteriol33:369-374, 1983). These indicate that the linkages of polypeptidoglycancell wall of strain 10R are most likely different from those of strainM58-1. Taxonomic analyses of cell wall indicate that there is a goodpossibility that strain 10R is a new species. More detailed taxonomicidentification for strain 10R was underway as of the date hereof.

ASSAY FOR PROTEOLYTIC ACTIVITY

The proteolytic activity in Nocardiopsis sp. cultures was determined bythe well known Anson hemoglobin method, cfr. Journal of GeneralPhysiology, 22, 79-89 (1959). One Anson unit is the amount ofproteolytic enzyme digesting hemoglobin at a pH value of 9.0 and atemperature of 25° C. during a reaction time of 10 minutes with such aninitial velocity that per minute there is formed such an amount of splitproducts which cannot be precipitated with trichloroacetic acid thatthese split products give the same color with phenol reagent as does onemilli-equivalent of tyrosine.

Proteolytic activity was also determined by the hydrolysis of casein andsubsequent reaction of TCA-soluble peptides with o-phthaldialdehyde and2-mercaptoethanol. The absorbance of the resultant complex is measuredat 340 nm and compared to a serine standard. Reaction mixtures arecomprised of 1.0 ml of 2.0% (W/V) Hammerstein casein and 0.5 ml of anappropriate enzyme dilution both in Universal Buffer I of Britton andRobinson, pH 9.5 (J. Chem. Soc. 1931, p. 1451). Mixtures are incubatedfor 30 minutes at 25° C. then the reaction terminated by addition of 2.5mls stop reagent (3.6% W/V trichloroacetic acid, 6.0% W/V sodium acetateand 3.78% v/v glacial acetic acid in deionized water). In controlreactions, the stop reagent is added prior to enzyme addition. After 20minutes at 25° C., the reaction mixtures are filtered through Whatmanfilter paper #42 or centrifuged.

An aliquot (200 ul) of the filtrate is added to 3 ml of OPA reagentcontaining 0.05M sodium tetraborate, 1% W/V sodium dodecylsulfate, 0.8mg/ml O-phthaldialdehyde (OPA) (originally dissolved as a 40 mg/mlsolution in ethanol), and 0.2% v/v 2-mercaptoethanol. After 2 minutes,the absorbance at 340 nm is determined. Similarly, a 200 ul aliquot of aserine standard (0.2 mg/ml) is added to 3 ml of OPA reagent and the A₃₄₀determined. Activity is expressed in CPU (Casein Protease Unit) where 1CPU is defined as the amount of enzyme which, under standard conditions,produces an amount of non-TCA-precipitable digestion products per minutecorresponding to 1 millimole of serine.

PREPARATION OF PROTEASE CONCENTRATE

The Nocardiopsis of the invention may be cultivated under aerobicconditions in a nutrient medium containing assimilable carbon andnitrogen together with other essential nutrients, the medium beingcomposed in accordance with the principles of the known art.

Suitable carbon sources are carbohydrates, such as sucrose, glucose, andmaltose, or carbohydrate containing materials such as cereal grains,malt, rice and sorghum. The carbohydrate concentration incorporated inthe medium may vary widely, e.g. 1 to 15%, but usually 8-10% will besuitable, the percentage being calculated as equivalents of glucose.

The nitrogen source in the nutrient medium should be of an organicnature. Among the organic nitrogen sources, quite a number are regularlyused in fermentation processes involving the cultivation ofactinomycetes. Illustrative examples are soybean meal, cotton seed meal,peanut meal, casein, corn steep liquor, yeast extract, and albumin. Inaddition, the nutrient medium should also contain the usual tracesubstances.

Since the Nocardiopsis strains of the invention are psychrophilic inthat they are unable to grow at temperatures above 35° C., thecultivation is preferably conducted in the temperature range of 20° C.to 30° C. and at alkaline pH values. The alkaline pH may be obtained byaddition of suitable buffers, such as sodium carbonate or mixtures ofsodium carbonate and sodium bicarbonate (after sterilization of thegrowth medium). For cultivation in tank fermentors, it is necessary touse artificial aeration. The rate of aeration may be that employed inconventional tank fermentation.

After fermentation, a liquid enzyme product may be produced by removalof coarse material from the broth and, if desired, through concentrationof the broth by evaporation at low temperature or by reverse osmosis.Finally, preservatives may be added to the concentrate.

According to the invention, alkaline protease can also be prepared bycultivation of a microorganism containing a gene encoding for andexpressing a protease derived from a Nocardiopsis strain of theinvention, followed by recovery of the protease from the culture broth.Said microorganism to be cultivated is either the Nocardiopsis strainitself (including mutants and variants), or is a transformed hostorganism wherein the gene has been inserted by recombinant DNAtechniques. Such techniques are known in the art and generally comprisethe following steps:

a) providing a suitable recombinant DNA cloning vector comprisingDNA-sequences encoding functions facilitating gene expression and aDNA-sequence encoding the Nocardiopsis protease;

b) transforming a suitable host organism with the cloning vector fromstep a); and

c) culturing the transformed host in a suitable culture medium andoptionally recovering the protease from the culture medium.

Preferred host organisms are strains of Nocardiopsis Streptomyces, yeastand Aspergillus. It is especially preferred to use A. oryzae as the hostaccording to EP 238,023 (Novo).

PEPTIDE MAPPING

The peptides produced by protease-catalyzed digestion of the oxidizedB-chain of insulin were separated by reverse phase HPLC. Chromatogramsof peptide digestion products obtained through the action of theNocardiopsis sp. strain 10R protease, purified as described in ExampleV, are shown in FIG. 1. For comparison, the peptides generated bydigestion with the known alkaline proteases, ALCALASE®, ESPERASE®,SAVINASE® are also given. It may be seen that the protease of thisinvention (10R) is distinctly different from any of the Bacillusalkaline proteases with regard to its pattern of digestion of theoxidized B-chain insulin. In addition, for strain 10R protease the peakwhich corresponds to intact B-chain insulin on the chromatogramdisappears at an earlier time point than with the Bacillus proteases.The protease from Nocardiopsis dassonvillei strain M58-1 produces adigestion pattern that is very similar to that of strain 10R (data notshown).

ENZYME PREPARATION

Solid enzyme preparations may be prepared from the purified and/orconcentrated broth by precipitation with salts such as Na₂ SO₄ or withwater miscible solvents such as ethanol or acetone. Removal of the waterin the broth by suitable drying methods such as spray drying may also beemployed. The proteolytic activity of protease preparations so obtainedis usually in the range of 0.2-1.0 AU/g. (approx. 0.2-1.0 CPU/g).

The protease preparation of the invention is preferably in a formsuitable for use as a detergent additive, particularly a non-dustinggranulate, a stabilized liquid or a protected enzyme.

Non-dusting granulates may be produced e.g. according to NL 167,993(Novo), U.S. Pat. Nos. 4,106,991 (Novo) or 4,661,452 (Novo) and mayoptionally be coated according to principles known in the art.

A liquid protease preparation may be stabilized e.g. by adding propyleneglycol, other polyols, sugars, sugar alcohols and boric acid. Otherenzyme stabilizers are known in the art.

Protected enzyme may be produced according to EP 238,216 (Novo, Albright& Wilson).

DETERGENT COMPOSITION

The detergent compositions of the invention comprise surfactant whichmay be of the anionic, non-ionic, cationic or zwitterionic type, or amixture of these. Typical examples of anionic surfactant are linearalkyl benzene sulfonate (LAS), alpha olefin sulfonate (AOS), alcoholethoxy sulfate (AES) and natural soap of alkali metals.

Detergent according to the invention may contain other detergentingredients known in the art, such as builders, bleaching agents, bleachactivators, anti-corrosion agents, sequestering agents, anti-soilredeposition agents, perfumes, stabilizers for the enzymes and bleachingagents and so on.

The detergent compositions of the invention can be formulated in anyconvenient form, such as powders, liquids, etc. The protease may bestabilized in a liquid detergent by inclusion of enzyme stabilizers,e.g. those mentioned above.

Detergents usually have a pH in solution of 7-12, especially 8-10.5. Dueto its broad pH optimum, protease of the invention is highly active inthis whole range.

The detergent of the invention may include one or more other detergentenzymes in addition to protease of the invention. Examples are lipase,amylase, cellulase and protease (other than protease of the invention),e.g. alkaline Bacillus protease. The two (or more) enzymes may be addedseparately or in the form of a combined additive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows elution chromatograms of digestion products of oxidizedB-chain of insulin with 10R protease of the invention. Similarchromatograms are shown for the undigested substrate and after digestionwith Savinase® and Alcalase® (prior-art proteases).

FIG. 2 shows temperature-activity and pH-activity curves for 10Rprotease of the invention.

FIGS. 3 and 4 show pH-activity and temperature-activity curves for twoproteases of the invention (10R and M58-1), compared with published datafor protease ZIMET 43647.

EXAMPLE I

Nocardiopsis sp. strain 10R was cultivated at 30° C. on a rotary shakingtable (250 rpm) in 250 ml triple-baffled Erlenmeyer flasks containing 50ml of medium of the following composition:

    ______________________________________                                        Maltodextrin M-100       20                                                   Soy bean flour           20                                                   Yeast Extract            2                                                    K.sub.2 HPO.sub.4        9                                                    CaCO.sub.3               5                                                    ______________________________________                                    

After sterilization, the pH of the medium was adjusted to 9.0 by theaddition of 5 ml of 1M solution of sodium carbonate/sodium bicarbonatebuffer, pH 9.2. After 2 to 5 days of incubation, the proteolyticactivity of the broth was determined using the Anson assay methoddescribed above. The enzyme activity of the 10R broth was 13.3 AU/1after 93.5 hours incubation.

Nocardiopsis dassonvillei strain M58-1 was cultivated as described,except that incubation was at 25° C. After 140.5 hours of incubation,the broth had an enzyme activity of 9.6 AU/1.

EXAMPLE II

Nocardiopsis sp. strain 10R was cultivated in the medium and underconditions as described in Example I . After 26 hours of growth, 2liters of the culture were used to inoculate 50 liters of the describedmedium in Example I in a fermentor, of which the pH of the medium wasnot controlled after sterilization.

After inoculation, the growth pH of the fermentor was controlled at 8.7with 2M Na₂ CO₃ and 10% H₃ PO₄. Temperature was maintained at 30° C.After 90 hours, protease titer was determined using the Anson assaymethod, a value of 11.5 Anson units/liter being indicated.

The culture broth was centrifuged to remove cells. Supernatant was thenfiltered (0.45μ) and concentrated by ultrafiltration (10,000 molecularweight cut-off) to become the product employed in the followingexamples.

Nocardiopsis dassonvillei strain M58-1 was cultivated in the medium andunder conditions as described in Example I, except the incubationtemperature was at 25° C. After 24 hours of growth, 2 liters of theculture were used to inoculate 50 liters of the fermentor mediumconsisting of the following components (grams/liter):

    ______________________________________                                        Maltodextrin           40                                                     Cerelose               40                                                     Soy bean flour         60                                                     K.sub.2 HPO.sub.4      5.5                                                    MgSO.sub.4             0.40                                                   CaCl.sub.2             0.30                                                   Trace mineral solution 7      ml                                              ______________________________________                                    

The temperature of the fermentation was controlled at 30° C. The initialpH was set at 8.6 and was controlled not to fall below 8.5 by additionof 2M Na₂ CO₃ and not to exceed 8.8 by addition of H₃ PO₄. A glucosefeed was started when glucose was depleted and the percent D.O. began torise. The feed stream consisted of a glucose solution (500 g cerelose in500 g H₂ O). The glucose was fed at a constant rate of 3.5 ml/liter/hruntil 20 g/liter glucose had been added (approximately 12 hours).

Protease began to accumulate at 40 hours with a peak titer of 53 AU/1obtained at 120 hours.

The culture broth was centrifuged to remove cells. Supernatant was thenfiltered (0.45μ) and concentrated by ultrafiltration (10,000 molecularweight cut-off) to become the product employed in the followingexamples.

EXAMPLE III

The protease activity of strain 10R from Example II as a function of pHand temperature was determined by using casein as the substrateaccording to the assay method described previously. In addition, theeffects of EDTA (10 mM) and phenylmethylsulfonyl fluoride (PMSF, 1 mM)on protease activity were examined. Results are depicted graphically inFIG. 2. As seen in FIG. 2 (a), the temperature optimum is greater thanor equal to 60° C., similar to the commercial protease Alcalase. Theprotease shows maximal activity between pH 8-9 and at least 85% of themaximum in the pH range 7-11. EDTA has no effect on protease activity,while the addition of PMSF results in almost complete inhibition,indicating that the protease is of the serine type.

EXAMPLE IV

The stability of proteases from strains 10R and M58-1 in the presence ofvarious detergent components at 25° C. or 40° C. was determined asfollows:

Proteases were diluted to 0.5 AU/1 in 0.01M borate buffer, pH 9.5 withor without the additional components shown in Table I. The samples wereincubated at 25° C. or 40° C. for 30 minutes, then assayed at 25° C. forprotease activity. Results are shown in Table I.

Both of the Nocardiopsis sp. proteases tested retained at least 83% oftheir original activities at either 25° C. or 40° C., thus making themsuitable for laundering use.

                                      TABLE I                                     __________________________________________________________________________    Stability of Proteases from Nocardiopsis sp. 10R and                          Nocardiopsis dassonvillei M58-1 in the Presence of Detergent                  Components                                                                    % Activity Remaining                                                          Incubation Conditions                                                         25° C.        40° C.                                                 no  10 mM                                                                             10 mM                                                                             0.1%                                                                              no  10 mM                                                                             10 mM                                                                             0.1%                                         Enzyme                                                                             add'n                                                                             CaCl.sub.2                                                                        EDTA                                                                              STPP*                                                                             add'n                                                                             CaCl.sub.2                                                                        EDTA                                                                              STPP                                         __________________________________________________________________________    10R  100  90  94 94  101  83  95  98                                          M58  100 102 102 98  105 110 105 111                                          __________________________________________________________________________     *STPP is sodium tripolyphosphate                                         

EXAMPLE V

The protease from Nocardiopsis sp. strain 10R prepared in Example II waspurified as follows:

Concentrated cell-free broth was first loaded onto a Sephadex-G25 column(medium grade) equilibrated in 0.05M HEPES, pH 7 buffer. Protein waseluted from the column in the same buffer and fractions containingprotease activity were pooled. If necessary, the pooled enzyme solutionwas diluted with water or buffer to give an appropriate ionic strength,then was loaded onto a CM-Sepharose Fast Flow column equilibrated in0.05M HEPES, pH 7. Approximately 50% of the total protease activitybound to the resin and was subsequently eluted with a linear saltgradient (0-75 mM NaCl) .

The CM-Sepharose purified protease possessed a specific activity of 21.0AU/g (15 CPU/g).

When analyzed by SDS-PAGE, the purified protease was shown to containtwo components, one major and one minor with molecular weights of 25,000and 23,500, respectively. Protease from Nocardiopsis dassonvillei strainM58-1 purified in a similar manner consisted of one band on SDS-PAGEwith molecular weight of 20,500.

                  TABLE II                                                        ______________________________________                                        Summary of Purification of Strain 10R Protease                                                          Total   Specific                                                                             Fold                                                   Total   Recovery                                                                              Activity                                                                             Puri-                                Step      AU/l    AU      (%)     (AU/g) fication                             ______________________________________                                        Crude     19.4    0.97    100     --     --                                   cell-free                                                                     concentrate                                                                   Sephadex-G25                                                                            16.8    0.89    92      6.2    1.0                                  CM-Sepharose                                                                            2.29    0.44    45      21.0   3.4                                  ______________________________________                                    

EXAMPLE VI

Proteases from Nocardiopsis sp. were distinguished from the commercialproteases ALCALASE®, SAVINASE®, and ESPERASE® by their pattern ofdigestion of the oxidized B-chain of insulin. Protease (2.7×10⁻⁵ CPU)was added to oxidized B-chain of insulin (0.17% w/v) in pH 9.0 bufferand incubated for 15 minutes or 2.5 hours at 25° C. After boiling 20minutes to quench the reactions, samples were injected onto areverse-phase HPLC column (Bakerbond Widepore C₁₈) equilibrated in 0.1Mammonium sulphate, pH 3. Peptides were eluted with a linear gradient of0-50% acetonitrile.

Chromatograms are shown in FIG. 1 and indicate that the protease of thisinvention is distinct from any of the Bacillus alkaline proteases withregard to its pattern of digestion of the oxidized insulin B-chain. Inaddition, for strain 10R protease, the peak which corresponds to intactB-chain insulin on the chromatogram disappears at an earlier time pointthan with the Bacillus proteases.

EXAMPLE VII

Washing tests were performed in a Terg-O-Tometer for ten minutes at 15°C. with EMPA 116 test fabric swatches (cotton soiled with blood, milk,and carbon black) supplied by Test Fabrics, Inc., Middlesex, N.J. Enzymedosages of 0.025, 0.05, 0.1 CPU/1 were added to Tide® (<0.5% phosphate,no enzymes, pH 10.4) . The proteases used were from strain 10R andSAVINASE®.

The cleaning ability of the proteases was measured by reflectance change(Δ R), i.e. the reflectance value of enzyme-washed test swatches minusthat of swatches washed without enzyme. Reflectance values were readwith the aid of a Gardiner Reflectometer XL 800 (Bethesda, Md.).

The results are shown in Table III. They indicate that it is possible toobtain 20-50% greater cleaning ability with 10R protease than withSavinase at the same dosage.

                  TABLE III                                                       ______________________________________                                        Wash Efficacy of 10R Protease and Savinase on EMPA 116                                            Reflectance Change                                        Enzyme Dose (CPU/l) (Δ R)   % Increase                                  ______________________________________                                        10R    0.025        3.9           50%                                                0.05         7.1           29%                                                0.10         11.5          20%                                         Savinase                                                                             0.025        2.6           --                                                 0.05         5.5           --                                                 0.10         9.6           --                                          ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Wash Efficacy of Nocardiopsis sp. Strain 10R and Nocardiopsis                 dassonvillei Strain M58-1 Proteases on EMPA 116, Spinach, and                 Blood Stains                                                                          Reflectance Change (Δ R)                                        Protease  EMPA 116      Spinach  Blood                                        ______________________________________                                        10R       7.3           4.7      8.2                                          M58-1     9.7           3.1      6.8                                          Savinase ®                                                                          5.7           3.5      2.8                                          Alcalase ®                                                                          2.7           3.1      5.5                                          ______________________________________                                    

EXAMPLE VIII

Washing tests were performed using proteases from Nocardiopsis sp.strain 10R and Nocardiopsis dassonvillei strain M58-1 and, forcomparison, the commercial proteases ALCALASE® and SAVINASE®. EMPA 116test fabric swatches (cotton soiled with blood, milk, and carbon black)were supplied by Test Fabrics, Ind., Middlesex, N.J.; spinach and bloodswatches were made in-house by applying fresh spinach extract or bovineblood to desired cotton fabric then air-drying. Wash tests wereperformed in a Terg-O-Tometer for ten minutes at 15° C. in a detergentof the following composition:

    ______________________________________                                        Component             g/l                                                     ______________________________________                                        LAS (Nansa 80 S, 80% active)                                                                        0.4                                                     AE (Berol 065)        0.15                                                    Soap (Sumlight, 80% active)                                                                         0.15                                                    Sodium tripolyphosphate                                                                             1.75                                                    Sodium silicate       0.4                                                     Carboxymethyl cellulose                                                                             0.05                                                    EDTA                  0.01                                                    Sodium sulfate        2.1                                                     Water                                                                         Adjusted to 9° dH German hardness, pH 9.5.                             ______________________________________                                    

An enzyme dosage of 0.05 CPU/1 was used with EMPA and spinach stains,0.5 CPU/1 with the blood stain.

Reflectance values were read with the aid of a Gardiner Reflectometer XL800 (EMPA and spinach) or an Elrepho 2000 at 460 nm (blood). Thecleaning ability of the proteases was measured by reflectance change (ΔR) , i.e. the reflectance value of enzyme-washed test swatches minusthat of swathes washed without enzyme. The results are compiled in TableIV.

It is evident from the results that the Nocardiopsis sp. strain 10R andNocardiopsis dassonvillei strain M58-1 proteases show equivalent tosuperior performance based on equal dosage on all types of stain tested.In particular, protease from strain M58-1 is the preferred enzyme foruse on EMPA 116, protease from strain 10R is preferred for use on bloodstains.

We claim:
 1. A detergent additive, comprising an alkaline serineprotease derived from a strain of Nocardiopsis, having a pH optimum ofabout 8 and retaining at least 60% of maximum activity at pH 7-11measured with casein as the substrate, said additive being in the formof a non-dusting granulate or a stabilized liquid.
 2. The detergentadditive according to claim 1, wherein the strain of Nocardiopsis is10R, M58-1, or a mutant thereof capable of producing the alkaline serineprotease.
 3. The detergent additive according to claim 2 having aproteolytic activity of 0.5-10 CPU/g.
 4. A detergent composition,comprising (a) an alkaline serine protease derived from a strain ofNocardiopsis, having a pH optimum of about 8 and retaining at least 60%of maximum activity at pH 7-11 measured with casein as the substrate and(b) a surfactant.
 5. The detergent composition according to claim 4having a proteolytic activity of 0.001-0.5 CPU/g.
 6. A process forpreparing an alkaline serine protease, comprising (a) cultivating astrain of Nocardiopsis aerobically under submerged conditions in anutrient solution containing suitable carbon and nitrogen sources at analkaline pH and a temperature in the range of 20°-30° C. and (b)recovering the alkaline serine protease from the culture broth, whereinthe alkaline serine protease has a pH optimum of about 8 and retains atleast 60% of maximum activity at pH 7-11 measured with casein as thesubstrate.
 7. The process according to claim 6, wherein the strain ofNocardiopsis is 10R, M58-1 or a mutant thereof capable of producing thealkaline serine protease.